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The P450-Glo™ Screening Systems provide a complete set of reagents for performing luminescent cytochrome P450 assays. The systems include a membrane preparation containing recombinant human cytochrome P450 enzyme, a luminogenic cytochrome P450 substrate appropriate for the enzyme, an NADPH Regeneration System, reaction buffer, Luciferin Detection Reagent and Luciferin-Free Water. The membranes are prepared from baculovirus-infected insect cells and contain human cytochrome P450 and P450 reductase (and cytochrome b5 for CYP2C9 and CYP3A4). The P450-Glo™ Screening Systems also contain a membrane fraction devoid of cytochrome P450 activity as a negative control. The assays are ideal for testing the effects of drugs and new chemical entities on cytochrome P450 enzyme activities.

The cytochrome P450 reaction is performed by incubating a luminogenic cytochrome P450 substrate with a cytochrome P450 enzyme and the NADPH Regeneration System. The luminogenic P450-Glo™ Substrates are derivatives of beetle luciferin ((4S)-4,5-dihydro-2-(6-hydroxybenzothiazolyl)-4-thiazolecarboxylic acid or d-luciferin), a substrate of firefly luciferase. The P450-Glo™ Substrates do not react with luciferase but are converted by cytochrome P450 to luciferin, which in turn reacts with luciferase to produce light. Light is used to monitor cytochrome P450 activity because the amount of light produced is directly proportional to the amount of d-luciferin produced by cytochrome P450.

The new P450-Glo™ CYP3A4 Screening System with Luciferin-IPA contains our latest 3A4 substrate and is the most sensitive P450-Glo™ 3A4 substrate available. Luciferin-IPA displays the widest range of inhibition profiles of all the P450-Glo™ CYP3A4 substrates.

Dimethyl sulfoxide (DMSO), a common solvent used to solubilize chemical compounds, can significantly inhibit the activity of the 3A4 isoform of cytochrome P450, even at low concentrations (<0.1%). The P450-Glo™ CYP3A4 Screening System (Luciferin-PPXE) DMSO-Tolerant Assay is specifically designed to tolerate DMSO in the 3A4 reaction. The assay exhibits little to no change in the signal-to-background ratio in the presence of 0.2% DMSO compared to a no-DMSO control.

After the cytochrome P450 reaction has been performed, the reconstituted Luciferin Detection Reagent is added. This reagent simultaneously stops the cytochrome P450 reaction and initiates a stable glow-type luminescent signal. The glow-type reaction produces a stable signal and eliminates the need for strictly timed luminescence detection. Protocols are configured for multiwell plate formats but can be easily adapted for single-tube applications.

Low False-Positive Rate: Use of a proprietary stabilized firefly luciferase and a proprietary luciferase assay formulation minimizes the incidence of false positives due to inhibition of luciferase by analytes when screening for cytochrome P450 inhibitors.

This product is available through the Promega Helix onsite stocking program in a –20°C Helix Freezer. The program offers numerous convenient solutions to meet your lab's needs. Helix Freezers are available in two sizes: 5.7 cubic ft. or 9.7 cubic ft.

Storage Conditions

Store the CYP1A2, CYP2C9 and CYP3A4 membranes at –70°C. Cytochrome P450 may lose activity with repeated freeze-thaw cycles. Avoid multiple freeze-thaw cycles by dispensing the CYP1A2, CYP2C9 and CYP3A4 membranes into single-use aliquots (e.g., 50μl for 96 reactions). Store aliquots at –70°C. All other components can be stored at –20°C or –70°C and protected from light. The reconstituted Luciferin Detection Reagent can be stored at –20°C for up to 3 months. For convenience, the reconstituted Luciferin Detection Reagent can be stored at room temperature (approximately 23°C) without loss of activity for 24 hours or at 4°C for 1 week. Avoid multiple freeze-thaw cycles of all components.

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